Interstitial Cells from Calcified and Healthy Aortic Valves Have Different Phenotype and Functions
Mariia Bogdanova1, Arsenii Zabirnyk1, Anna Malashicheva2, Katarina Zihlavnikova Enayati1, Kåre-Olav Stensløkken1, Arkady Rutkovskiy3, Jarle Vaage3.
1Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway, 2Almazov National Medical Research Centre, Saint-Petersburg, Russian Federation, 3Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
OBJECTIVE: Valve interstitial cells (VICs) are crucial for the development of calcific aortic valve disease. We aimed to compare the phenotype and functions of VICs from calcified and healthy aortic valve.
METHODS: VICs were isolated from human calcified and healthy aortic valves sampled during aortic valve replacement or transplantation. To induce calcification VICs were cultured 21 days in osteogenic medium. Expression of osteogenic marker bone morphogenetic protein 2 (BMP2) was analyzed by RT-qPCR, calcification was quantified using Alizarin Red staining. To differentiate VICs into myofibroblasts we stimulated them with TGFβ1. The myofibroblastic markers alpha-smooth muscle actin (αSMA) and calponin was measured by FACS and immunostaining. For adipogenic differentiation cells were stimulated with adipogenic medium and the expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG) and lipoprotein lipase (LPL) was analyzed by RT-qPCR. The expression of stem cell-associated marker CD106 and aldehyde dehydrogenase activity were measured by FACS. Cell proliferation was manually counted in Bürker chamber.
RESULTS: Unlike VICs from healthy valves, VICs from calcified valves cultured for 21 days without osteogenic stimulation stained positively with Alizarin Red confirming their calcific phenotype. Stimulation with osteogenic medium increased calcification and expression of BMP2 in both cells from calcified and healthy valves. Upon stimulation with myofibroblastic or adipogenic medium VICs from calcified valves had lower expression of myofibroblastic (p=0.0001 for αSMA, p=0.03 for calponin) and adipogenic (p=0.007 for PPARG, p=0.03 for LPL) markers than VICs from healthy valves. VICs from calcified valves had lower expression of stem-cell associated markers CD106 (p=0.04) and aldehyde dehydrogenase (p=0.04) suggesting their lower “stemness” degree. Cells from healthy valves proliferated faster than cells from calcified valves.
CONCLUSIONS: VICs from calcified aortic valves have a pro-osteogenic phenotype with less ability for proliferation and myofibroblastic differentiation as well as reduced multipotency compared to VICs from healthy valves.
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